1. Field of the Invention
The present invention generally relates to diagnostic devices, more specifically to non-instrumented biochemical diagnostic devices.
2. Background Art
There are numerous chemical and biological processes and diagnostic methods that require precisely controlled temperature conditions. One prominent example is the polymerase chain reaction (PCR), which allows for the specific amplification of a deoxyribonucleic acid (DNA) sequence from undetectable, small copy numbers to greater, detectable copy numbers. The ability to amplify DNA to detectable levels has made PCR an essential tool for diagnostics.
PCR requires multiple repetitions of heat cycles typically ranging from 50° C. to 95° C. A 95° C. incubation of the reaction mixture ensures melting of double stranded DNA, a drop in temperature to 50° C. allows annealing of primer sequences to the target DNA sequences, and subsequent extension by DNA polymerase at approximately 72° C. For a typical diagnostic 100-300 bp amplicon using a DNA polymerase with a polymerization rate of 20-100 bp/sec, the major limiting step is the time required to heat and cool the reaction mixture fluid to temperature for each cycle. Presently, energetically and technologically expensive thermal cyclers are required for this process. However, such thermal cyclers are prohibitive for many applications such as remote surveillance studies and diagnostics in clinical settings with limited resources, for example, in developing countries. There is a need in the art, therefore, for a simple, non-instrumented means that can provide output temperatures within relatively precise tolerances without the assistance of other equipment.
There are several other approaches for amplifying nucleic acid signal. The more compelling of which are isothermal. In one such example: Nucleic acid sequenced based amplification (NASBA), nucleic acid signal amplification is performed by amplification of RNA using three enzymes reverse-transcriptase, RNase H and T7 RNA polymerase. Combined with the right primers these enzymes can amplify RNA signal under isothermal conditions. Other examples of isothermal nucleic acid signal amplification are (but not limited to): transcription mediated amplification (TMA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), and helicase dependent amplification (HDA). For all these technologies there is a need in the art, therefore, for a simple, non-instrumented means that can provide output temperatures within relatively precise tolerances without the assistance of other equipment.
Another example of a biological process useful in the field of molecular diagnostics is reverse-transcription to generate complimentary DNA (cDNA) from RNA. The enzyme reverse-transcriptase generates cDNA from RNA by extension of a DNA primer which is annealed to RNA oligonuceoltides. This reaction is typically performed in vitro between 37° C. and 55° C. cDNA is more stable than RNA. A device that can maintain temperature between 37° C. and 55° C. for 30 minutes or more would facilitate generation of cDNA in clinical settings with limited resources as means to stabilize the RNA signal.